Study On The Role Of HMOF-Mediated YY1 Acetylation In Colon Cancer Cell Proliferation

MOF is a member of the MYST family of histone acetyltransferases and is highly conserved from Drosophila to human.Human MOF can form two different complexes in cells-MSL and NSL,both of which can acetylate H4K16,while NSL can also acetylate H4K5 and H4K8.In addition to altering chromatin structure through acetylation modifications and thus playing an important role in gene transcription regulation,MOF and its complexes can also participate in the regulation of various biological processes in cells,including tumorigenesis and development,through posttranslational modifications of non-histone proteins.In addition to the important role in gene transcription regulation through acetylation,MOF and its complexes can also participate in the regulation of various cellular biological processes,including tumor development,through the post-translational modification of non-histone proteins,but the detailed molecular mechanisms are not yet clear.YY1 is a well-known zinc finger protein transcription factor with dual regulatory roles of gene transcriptional activation and transcriptional repression.According to research reports,high level expression of YY1 in tumor tissues is closely related to the processes of proliferation,invasion,and migration of cancer cells.Colon cancer is a relatively common malignant tumor,which has been a serious threat to human health so far due to its high incidence and lethality.However,the specific mechanism of YY1 in colon cancer,especially the correlation with MOF,has not been reported.We found that MOF was enriched in the YY1 promoter by ChIP-Seq genomics analysis in the previous stage,and confirmed that the NSL complex promoted cell proliferation by increasing the expression level of YY1 protein in hepatocellular carcinoma cells,suggesting that MOF has a regulatory effect on the expression of YY1,but the specific mechanism of the interaction between the two,as well as whether MOF has an acetylation modification of YY1,has yet to be deeply investigated.However,the specific mechanism between the two and the acetylation modification of YY1 by MOF need to be investigated in depth.In this paper,we investigated the role and molecular mechanism of human MOF-mediated acetylation modification of YY1 in colon cancer proliferation.The study is summarized as follows:First,in colon cancer cells,MOF(or MSL complex)was confirmed to interact with YY1 by immunoprecipitation and protein immunoblotting experiments;subsequently,using different truncated somatic plasmids of YY1 and MOF proteins capable of being expressed in prokaryotic and mammalian cells as well as proteinprotein interbinding experiments,it was demonstrated that the C-terminal end of MOF binds directly to 146-270 aa of YY1;Meanwhile,it was found that the MOF/MSL complex could acetylate YY1 and the modification promoted the ubiquitination degradation of YY1,and it was further clarified that after the deletion of 146-270 aa of YY1,MOF no longer affects its ubiquitination level,which suggests that this region is the key region of YY1 acetylation-ubiquitination modification.Secondly,potential ubiquitination modification sites K183/K208/K258 in the region of 146-270 aa were predicted through bioinformatics websites and review of the literature.using the constructed corresponding point mutant plasmids in combination with MOF and HDAC inhibitors,K183/K258 were identified as the key sites for ubiquitination modification of YY1,and it was found that YY1 acetylation modification modulates K183-mediated ubiquitination.Again,the reporter gene plasmid of pp53-TA-Luc luciferase inserted with multiple p53 response elements was chosen as a tool to analyze the regulatory effect of MOFYY1 on p53-mediated transcriptional activity by detecting its dual luciferase activity.The experimental results showed that MOF-mediated acetylation-ubiquitination of YY1 attenuated the inhibitory effect of YY1 on p53 transcriptional activity,suggesting that intracellularly MOF-YY1 may be synergistically involved in the regulation of transcriptional activity towards p53.In addition,plate colony formation assay demonstrated that MOF significantly inhibited the ability of YY1 to promote colony formation in HCT116 colon cancer cells,suggesting that MOF-YY1 is synergistically involved in regulating the clone formation process.Finally,the effect of the K183 site of YY1 on the transcriptional activation of the CDC6 gene was examined using a reporter gene plasmid of pGL3-CDC6-Luc luciferase containing sequences from the CDC6 promoter region(which contains two YY1 binding motifs).The experimental results showed that the K183 locus of mutant YY1 significantly inhibited the transcriptional activation of CDC6 gene by YY1.In addition,the proliferation assay,MTT assay and plate colony formation assay of Ed U-doped cells confirmed that the K183 locus of mutant YY1 antagonized the promotional effect of YY1 on the proliferation of colon cancer cells,suggesting that K183 associated with the YY1 acetylation-ubiquitination modification site associated with YY1 acetylationubiquitination modification,suggesting that the K183 locus plays an important role in the proliferation of colon cancer cells.In summary,through a series of biological experimental studies,this thesis demonstrated for the first time that the acetyltransferase MOF(or MSL complex)directly binds to and acetylates YY1,which in turn promotes the ubiquitination degradation of YY1,and further proved that acetylation-ubiquitination modification of YY1 inhibits the proliferation of colon cancer cells through the promotion of p53-mediated transcriptional activity.The key amino acid site of YY1 in the MOF-YY1 binding region and the MOF-mediated acetylation-ubiquitination pathway was identified as K183;this further validated the important role of the K183 site in the transcriptional regulation of YY1 and its role in the proliferation of colon cancer cells.In conclusion,our study reveals that YY1 is a novel substrate for the catalytic action of the MOF/MSL complex acetyltransferase,and the two may be synergistically involved in the regulation of colon cancer cell proliferation through the acetylationubiquitination mode in the cell.This study expands the theory of new molecular mechanism of colon cancer development,and will provide new potential therapeutic targets and theoretical basis for targeted therapy of colon cancer.